Characterization of naturally occurring murine cytotoxic cells has been further developed to understand the origin, differentiation and normal function of this population. Cells from spleen, thumus, blood, bone marrow and liver have been characterized. Effector cell activity has been monitored against appropriate targets for both natural killer (NK) activity and natural cytotoxic (NC) activity. Phenotype has been determined by complement (CO-mediated, antibody-dependent cytotoxicity, by flow cytometry analysis (FCA) of immunofluroescence (IF) and by visual morphological assessment using Staphylococcus aureus protein A-dependent (SpA) binding to monoclonal antibody (MoAb). Large granular lymphocytes (LGL) from nylon wool nonadherent cells subjected to density separation techniques have been characterized with a series of MoAb to T-cell differentiation antigens, to myelomonocytic antigens and to other hematopoietic subsets. Initially we examined liver-derived LGL from animals whose NK activity has been augmented biological response modifiers (BRM). In addition, LGL obtained from livers of BRM-treated mice have been compared to splenic subpopulations enriched for LGL. Purification procedures for splenic LGL from normal and BRM-activated animals required modification and additional steps to eliminate low density T cells (Lyt2+ or L3T4+) and B cells. Pronounced differences in phenotype between the liver derived and spleen-derived LGL populations has been observed. The phenotypic differences do not appear to be related to the BRM used for NK augmentation but to the organ used as a source of LGLs. We have currently begun experiments to select lymphocyte subsets before culture in lymphokine to identify the lymphokine activated killer (LAK0 cells and to compare these cells with NK cells and T cells. Characterization of bone marrow progenitors of NK activity has been initiated to determine whether the precursors share any of the markers found on mature, functional NK cells.